The overall objective of our research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Our general strategy is to focus on an evolutionarily conserved protein complex, SWI/SNF, which is required for expression of a subset of yeast genes and for the activity of transcriptional activators. Genetic studies in yeast indicate that SWI/SNF facilitates transcription by antagonizing chromatin-mediated transcriptional repression, and our in vitro studies indicate that the ~1Mda SWI/SNF complex can use the energy derived from ATP hydrolysis to mobilize nucleosomes and disrupt nucleosome structure. SWI/SNF complexes are essential for mammalian development and inactivation SWI/SNF subunits lead to cancers in humans. This proposal continues to exploit the powerful genetic and biochemical opportunities available in yeast to investigate the role of SWI/SNF in vivo and the biochemical mechanism by which SWI/SNF disrupts nucleosome structure in vitro. The first aim of this proposal will test the hypothesis that the SWI/SNF and ISWI remodeling complexes function antagonistically at many inducible genes. This aim is addressed by chromatin immunoprecipitation assays and nucleosome mapping methods. The second objective will test the hypothesis that SWI/SNF facilitates transcription by disrupting the higher order folding of chromatin. These studies will involve sedimentation velocity analyses of remodeled nucleosomes as well as biochemical analyses of Sin-histones. Aim 3 describes single molecule assays and SWI/SNF-DNA photo-affinity crosslinking studies to dissect the role of ATP hydrolysis in chromatin remodeling activity. The fourth aim will address the structure of SWI/SNF bound to a mononucleosome using single particle reconstruction of cryo-EM images. This aim also investigates the subunit-subunit interactions required for assembly of SWI/SNF. This proposal describes research that is focused on how chromosome structure affects gene expression and how the normal cellular machinery contends with this structure. Specifically, we propose continued studies on a novel protein machine, the SWI/SNF complex that facilitates gene activation by remodeling chromosome structure. This machine is essential for mammalian development and inactivation of human SWI/SNF leads to a variety of cancers.